Most human malignancies will respond to current chemotherapeutic treatment regimens, yet few neoplasms can be cured by chemotherapy alone. Development of more effective drugs is thus necessary, but we believe that a high priority should also be placed on optimizing the use of available agents, which in turn requires a better understanding of the factors which modulate drug activity in a tumor. While drug 'resistance' is generally implicated as the cause of treatment failure, it is seldom possible to know when, where, or why resistant cells appear in the tumor mass. This information can, however, be determined in certain laboratory models, like V79 multicell spheroids in tissue culture. We thus intend to evaluate several clinically-used chemotherapeutic agents in spheroids, emphasizing regional and temporal variations in cytotoxicity; our results will suggest ways to use each agent more effectively, and provide additional criteria for selecting (and using) drug combinations. Regional variations in drug efficacy will be studied in spheroids stained with slowly-penetrating, non-toxic fluorescent dyes. Since the dyes are retained in disaggregated cells, a fluorescence- activated cell sorter can be used to select subpopulations with specified stain intensities; the clonogenicity of these sorted cells then defines the drug toxicity throughout the spheroid. The location, timing and degree of resistance to each drug will suggest useful combinations, as well as indicating ways to improve single- agent efficacy. In subsequent multidrug studies, therapeutically useful interactions, including differential (complementary) toxicity, reoxygenation, cell cycle perturbations, metabolic alterations, and/or direct interaction of lesions, will be identified and quantified. The resulting information will supplement that already available for these clinically effective drugs, so promising combinations, doses and sequences and can be evaluated in patients without delay.